Study of hereditary prostate cancer and human artificial chromosomes

Division Of Basic Sciences - Nci

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Abstract

Genetic linkage studies implicate a gene or genes at Xq27 in hereditary prostate cancer susceptibility (HPCX). The corresponding region spans 750 kb and includes five SPANX genes (SPANX-A1, -A2, -B, -C, and D), which encode proteins that are expressed in sperm nuclei and a variety of cancer cells. Each SPANX gene is embedded in a recently-formed segmental duplication (SD) up to 100 kb in size, resulting in extensive enrichment in long stretches of repeated DNA in this region. Due to their recent amplification, both SPANX coding and flanking sequences in SDs are nearly identical throughout the SPANX-A/D cluster, which complicates mutational analysis of these genes by PCR. However, we succeeded in analysis of SPANX genes from prostate cancer patients, using TAR cloning technique, which makes it possible to directly isolate large genomic segments from complex genomes. This analysis revealed frequent gene deletion/duplication and homology-based sequence transfers involving SPANX genes at Xq27, suggesting that SD-mediated homologous recombination in this region might be a source for predisposition to hereditary prostate cancer. In our recent work, a search for large genomic rearrangements at Xq27 was undertaken using three-color FISH. This analysis was carried out with several extensively studied families with X-linked hereditary prostate cancer. Inversion of the chromosomal region including the SPANX gene cluster was found in affected but not in unaffected brothers. Thus our results are consistent with the hypothesis that this inversion is causally-related to susceptibility to prostate cancer in the affected patients. However, the molecular basis of such causal relationship is not known yet. We hypothesize that the inversion leads to activation of expression of SPANX proteins that may play a role in cancer progression in multiple human cell types. During the past year, we have concentrated on mapping the breakpoint(s) of the inversion. This analysis was performed using a combination of Southern blot-hybridization, PCR, TAR cloning and DNA sequencing. Several candidate breakpoint regions have been identified within 1 Mb genomic sequence corresponding to SPANX gene cluster and flanking sequences. Verification of these candidate regions is in progress. A future work will focus on physical mapping of breakpoint(s) and analysis how the inversion alters expression of genes at/near breakpoints which could lead to malignancy. After more than two decades of investigation, human centromeres remain enigmatic and poorly understood. Some progress in this field was outlined after demonstration by Hunt Willards group that alpha satellite DNA (alphoid DNA), the primary DNA found in human centromeres, can induce the seeding of a kinetochore complex in human HT1080 cells. Several groups have confirmed this observation and reported the formation of Human Artificial Chromosomes (HACs) in human cells, using a transfection strategy that involved alpha satellite DNA. These HACs are maintained as single copy episomes in the nucleus and have a fully functional kinetochore. The development and detailed studies of HACs offer new approaches for: 1) elucidating the mechanisms for de novo centromere/kinetochore formation and its structural/functional organization, and 2) development of gene delivery vectors with potential therapeutic applications. The role of chromatin structure in kinetochore function has been studied intensively but still remains poorly understood. Recently we have generated a HAC in human HT1080 cells with a conditional centromere, which we expect to be instrumental in resolving many questions. The HAC includes approximately 6,000 copies of the tetracycline operator (tet-O) sequence. Such configuration allows a specific manipulation of the protein complement of a single kinetochore in vivo by targetting with tet-R fusion proteins. This approach has been used to target chromatin modifying proteins into the HAC and to demonstrate that a balance between open and condensed chromatin is critical for kinetochore function. The strongest effect on the synthetic kinetochore was observed after targeting of transcriptional repressors inducing HP1alpha-repressive chromatin. Our collaborative studies with William Earnshaws laboratory showed that the disruption of kinetochore structure by a transcriptional repressor reflects a hierarchical disassembly of kinetochore components reflecting a pattern of protein interactions within kinetochore. During the past year, we demonstrated that H3K4me2 chromatin domains are essential for maintaining centromere integrity. These results revealed a functional link between transcription potential of alphoid DNA and maintenance of the centrochromatin signature. This observation provides more evidence that centrochromatin resembles chromatin domains specific for actively transcribed genes. In future studies, our system will be used to investigate the structural/functional organization of the human kinetochore. Until recently, HACs were primarily generated and analyzed in HT1080 human cells. No HAC formation was observed in HeLa, BJ1 and TIG-7 cells. Our studies indicate that in these cells centromeric chromatin is highly enriched in H3K9me3 and HP1alpha compared to HT1080 cells. This suggests that differences in centromere heterochromatization may influence HAC formation and/or its stability in human cells. Indeed in our recent studies, we demonstrate that tethering of the histone trimethylase Suv39h1/KMT1A negatively regulates de novo CENP-A chromatin assembly and HAC formation on transfected alphoid DNAs carrying multiple tet-O sequences in HT1080, whereas tethering of histone acetyl-transferases p300/KAT3B or PCAF/KAT2B positively regulates HAC formation and kinetochore establishment in HeLa cells. These results indicate that chromatin assembly balance on alphoid DNA in the individual cell line is crucial for the de novo kinetochore assembly and that such an epigenetic barrier can be modulated. Practically, it means that de novo HAC formation may be induced in any type of human cells using alphoid DNA constructs with multiple tet-O sequences after their tethering by chromatin modifiers fused to the tet-repressor. HACs which carry a fully functional centromere are not associated with random mutagenesis and offer greater control over expression of ectopic genes. To adopt our HAC with a conditional centromere for gene delivery and gene expression in human cells, a loxP cassette was inserted into the HAC by homologous recombination in chicken DT40 cells following microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into chinese hamster ovary (CHO) cells, and a set of transgenes with the size up to 100 kb was efficiently and accurately incorporated into the tetO-HAC vector. The EGFP transgene (20 kb) was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted into the tet-O HAC can be eliminated from the cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy. Our future work will focus on expression of full-size human genes, VHL, NBS1, BRCA1, and ATM, in the HAC. After insertion of these genes into the HAC, it will be transferred into gene-deficient human cell lines via MMCT for complementation analysis. In addition, the tet-O-HAC will be tested for generation of iPS cells after insertion of the OCT4, SOX2, KLF4, cMYC cassette. Use of the HAC with a conditional centromere provides opportunity to eliminate the HAC along with the stem cell-inducing factors from the cell that allows to avoid insertional mutagenesis and cell transformation, complications that are frequently observed during cell re-programming.